Anti-tumor activities and apoptotic mechanism of ribosome-inactivating proteins

Ribosome-inactivating proteins(RIPs) belong to a family of enzymes that attack eukaryotic ribosomes and potently inhibit cellular protein synthesis.RIPs possess several biomedical properties,including anti-viral and anti-tumor activities.Multiple RIPs are known to inhibit tumor cell proliferation through inducing apoptosis in a variety of cancers,such as breast cancer,leukemia/lymphoma,and hepatoma.This review focuses on the anti-tumor activities of RIPs and their apoptotic effects through three closely related pathways:mitochondrial,death receptor,and endoplasmic reticulum pathways.

Ribosome Inactivating Proteins from Plants Inhibiting Viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

Crystallization and Preliminary Crystallographic Studies of Cucurmosin 2,a Ribosome-inactivating Protein from the Sarcocarp of Cucurbita moschata

Cucurmosin 2, a type 1 ribosome-inactivating protein （RIP） isolated from sarcocarp of Cucurbita moschata, has been crystallized by the vapor-diffusion method using PEG6000 as the precipitant. The crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 55.853, b = 65.507, c = 91.754 А, and have one molecule per asymmetric unit. X-ray data have been collected to 1.8А, using a synchrotron source.

Crystallization and Preliminary Crystallographic Studies of Luffaculin 1,a Ribosome-inactivating Protein from the Seeds of Luffa Acutangula

Luffaculin 1 purified from the seeds of Luffa acutangula belongs to the type I ribosome-inactivating proteins （RIPs）. It has been crystallized by the vapor-diffusion method using polyethylene glycol as a precipitant. The crystal is of space group P1 with a = 39.135, b = 46.813, c = 83.571 A, α = 891068,β = 80.009 and y = 72.143°, and has two molecules per asymmetric unit. X-ray data have been collected to be 1.4 A using a synchrotron source.

Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of-29 kD. It is a rRNA Nglycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a ICs0 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

Isolation of a genomic DNA for Jatropha curcas ribosome inactivating protein and its tobacco transformation

Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector pBI121-JRIP was constructed by inserting the JRIP gene into pBI121 plasmid. The recombinant Agrobacterium EHA105 strain harboring pBI121-JRIP was constructed by conducting pBI121-JRIP to strain EHA 105. PCR and Southern blotting were carried out, and the results proved that the JRIP gene was integrated into tobacco genome. It might provide a new material for disease resistance tobacco species breeding.

Visualization of interaction between ribosome-inactivating proteins and supercoiled DNA with an atomic force microscope

The interaction between ribosome-inactivating proteins (RIPs) and supercoiled DNA was observed with an atomic force microscope (AFM). It was found that RIPs can bind to both supercoiled DNA and the unwound double stranded loop region in supercoiled DNA. The RIPs hound to the supercoils can induce the conformational change of supercoiled DNA. Furthermore, the supercoiled DNA was relaxed and cleaved into nick or linear form by RIPs. It indicated that RIP seemed to be a supercoil-dependent DNA binding protein and exhibited the activity of su-percoil-dependent DNA endonuclease.

全蛋液微波巴氏杀菌动力学及蛋白质变化研究

热传导式的传统巴氏杀菌方法由于传热方式的限制,会导致全蛋液的过度热加工,造成蛋白质变性、产品品质劣变。微波体积式加热效率高,有利于降低巴氏杀菌过程对热敏蛋白质的影响。该文研究了全蛋液的微波巴氏杀菌,探究了微波巴氏杀菌对全蛋液中3种典型微生物(大肠杆菌、沙门氏菌和金黄色葡萄球菌)的杀菌效率、微波巴氏杀菌动力学及对全蛋液蛋白质的影响。结果表明,微波巴氏杀菌升温迅速(<50 s)且杀菌效果显著(致死对数>5 log10)。Log-Logistic模型拟合精度和稳定性最佳(R2:0.998~0.999,均方根误差:0.002~0.072),Log-linear+Tail模型拟合效果好且变量少(R2:0.982~0.999,均方根误差:0.026~0.298)。模型拟合结果与存活曲线趋势一致,进一步证明了模型的有效性和准确性。微波巴氏杀菌使蛋白质溶解度增加0.021~0.030。微波诱导蛋白质适度展开,极性基团暴露,游离巯基含量增加,有利于产品品质的改善。

绵羊源荚膜血清D型多杀性巴氏杆菌的生物学特性及2种候选疫苗的免疫效果评价

旨在研究新疆绵羊源荚膜血清D型多杀性巴氏杆菌(PmD)的生物学特性,探索其灭活疫苗与OmpH重组疫苗对小鼠的免疫保护效果,为防控新疆地区绵羊巴氏杆菌病提供参考。采用细菌分离鉴定、PCR鉴定、药敏试验、耐药与毒力基因检测、致病性试验等方法初步明确PmD的生物学特性;并通过最佳灭活条件和佐剂筛选、安全性试验、原核表达等方法制备PmD灭活疫苗与PmD-OmpH重组疫苗,采取小鼠攻毒保护试验评价2种疫苗的免疫效力。结果:从绵羊肺脏中分离出1株PmD,药敏试验显示,该菌为7重耐药菌,未检测到耐药基因;毒力基因检测显示,该菌具有12种毒力基因(exbB、exbD、hgbA、tonB、fimA、Oma87、OmpH、Psl、sodA、sodC、tbpA、toxA);致病性较强,对小鼠的最小致死菌量为2.2×105 CFU;灭活疫苗和重组疫苗对相同血清型菌株攻毒的保护率分别为80%和10%;灭活疫苗对血清型A、F和未知菌株攻毒保护率分别为0、20%和0,而重组疫苗的保护率则全为0。本研究发现1株强耐药、强毒力的绵羊源PmD,灭活疫苗对同种血清型菌株的保护性远高于重组疫苗,发病地区羊场可采用同种血清型灭活疫苗进行预防和控制绵羊巴氏杆菌病,对多价疫苗和新型疫苗的研制还有待进一步发掘。

国内抗富亮氨酸胶质瘤灭活蛋白1抗体脑炎的临床特征分析

目的探讨国内抗富亮氨酸胶质瘤灭活蛋白1(LGI1)抗体脑炎的临床特征、治疗及预后。方法在回顾我院1例确诊为抗LGI1抗体脑炎患者病历资料的基础上,对中国生物医学文献数据库、中国知网及万方数据库检索获得的2013—2023年国内核心期刊公开报道确诊的452例抗LGI1抗体脑炎患者的临床资料进行汇总分析。结果抗LGI1抗体脑炎患者的首发症状发生率最高的是癫痫发作,依次为记忆力减退、精神行为异常及其他症状;主要症状中的癫痫发作以面臂肌张力障碍发作(FBDS)、部分性发作及强直阵挛发作为表现。部分患者合并低钠血症。MRI主要显示颞叶内侧、海马、基底神经节和岛叶异常信号。大多数患者的血清和/或脑脊液LGI1抗体检测呈阳性。多数患者免疫球蛋白和/或类固醇治疗有效,少部分需要长期口服免疫抑制剂,一般预后良好。结论抗LGI1抗体脑炎最常见症状是癫痫发作、记忆力减退以及精神行为异常。血清抗LGI1抗体阳性率高于脑脊液。脑MRI多为颞叶异常信号。尽管部分患者可能在短期内复发,但经免疫治疗一般预后良好。

Dysregulation of mTOR activity through LKB1 inactivation

Mammalian target of rapamycin (mTOR) is aberrantly activated in many cancer types, and two rapamycin derivatives are currently approved by the Food and Drug Administration (FDA) of the United States for treating renal cell carcinoma. Mechanistically, mTOR is hyperactivated in human cancers either due to the genetic activation of its upstream activating signaling pathways or the genetic inactivation of its negative regulators. The tumor suppressor liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is involved in cell polarity, cell detachment and adhesion, tumor metastasis, and energetic stress response. A key role of LKB1 is to negatively regulate the activity of mTOR complex 1 (mTORC1). This review summarizes the molecular basis of this negative interaction and recent research progress in this area.

Trichobitacin-a new ribosome-inactivating protein Ⅰ.The isolation,physicochemical and biological properties of trichobitacin

From the press-residue of the fresh root tuber of Trichosanthes kirilowii Maxim (Cu-curbitaceae),a new ribosome-inactivating protein (RIP),trichobitacin,was isolated.It has the activity of RNA N-glycosidase and can inhibit the growth of human placental trophoblastic cells.Its molecular weight is 27,228 Da (ES-MS) and pI 9.6.It is a single chain basic RIP.Its amino acid composition was determined.It is a new RIP.It consists of 0.7~0.9% galactose and may be a glycoprotein.Its A'-and C-terminal amino acid is Asp and Ala,respectively.Its N-terminal preliminary amino acid sequence has been determined.

Induction of Apoptosis in Hormone-resistant Human Prostate Cancer PC3 Cells by Inactivated Sendai Virus

Objective Inactivated Sendai virus particle [hemagglutinating virus of Japan envelope （HVJ-E）] has a potential oncolytic effect due to its ability to induce apoptosis in tumor cells. However, the molecular mechanism of apoptosis induction in cancer cells mediated by HVJ-E has not been fully elucidated. This paper aims to investigate the underlying mechanism of apoptosis induction by HVJ-E in prostate cancer cells （PC3）. Methods PC3 cells were treated with HVJ-E at various MOI, and then interferon-β（IFN-β） production, and the cell viability and apoptosis were detected by ELISA, MTl--based assay and flow cytometry, respectively. Next, the roles of Jak-Stat, MAPK and Akt pathways played in HVJ-E-induced apoptosis in PC3 cells were analyzed by immunoblot assay. To further evaluate the cytotoxic effect of HVJ-E on PC3 cells, HVJ-E was intratumorally injected into prostate cancers on BALB/c-nude mice, and the tumor volume was monitored for 36 days. Results HVJ-E induced iFN-β production and activated Jak-Stat signaling pathway, which resulted in the activation of caspase-8, caspase-3, and PARP in PC3 prostate cancer cells post HVJ-E treatment. Furthermore, we observed for the first time that p38 and Jnk MAPKs in PC3 cells contributed to HVJ-E-induced apoptosis. In addition, intratumoral HVJ-E treatment displayed a direct inhibitory effect in an in vivo BALB/c nude mouse prostate cancer model. Conclusion Our findings have provided novel insights into the underlying mechanisms by which HVJ-E induces apoptosis in tumor cells.

抗富亮氨酸胶质瘤失活蛋白1抗体和抗接触蛋白相关蛋白2抗体脑炎28例临床特征

目的:抗富亮氨酸胶质瘤失活蛋白1(leucine-rich glioma-inactivated protein 1,LGI1)抗体、抗接触蛋白相关蛋白2(contactin-associated protein-like 2,CASPR2)抗体引起的自身免疫性脑炎较为罕见,临床异质性大,极易漏诊、误诊。本研究旨在总结抗LGI1抗体及抗CASPR2抗体脑炎患者的临床特征。方法:收集2018年1月至2021年1月中南大学湘雅医院神经内科收治的抗LGI1抗体、抗CASPR2抗体脑炎患者共28例,所有患者进行自身免疫性脑炎相关抗体谱及副肿瘤抗原谱抗体筛查。回顾性分析28例患者的临床表现、辅助检查资料、治疗及随访情况,采用改良Rankin量表评估患者治疗前后的生活能力,并对这2种自身免疫性脑炎的临床特征进行比较和分析。结果:28例中抗LGI1抗体脑炎17例,发病年龄28~83(53.18±19.08)岁,男女患者之比为9꞉8。临床表现为认知功能障碍的患者有10例,癫痫发作7例,面臂肌张力障碍发作4例,精神行为异常1例。实验室检查发现低钠血症7例;脑脊液检查异常4例,表现为白细胞数或蛋白质含量轻度上升各2例;脑电图异常13例,主要表现为慢波增多(8/13)及痫性放电(5/13);颅脑磁共振成像(magnetic resonance imaging,MRI)异常13例,主要表现为颞叶、海马及基底节区T2加权成像(T_(2)-weighted imaging,T_(2)WI)及水抑制反转恢复序列(fluid attenuated inversion recovery,FLAIR)呈高信号。抗CASPR2抗体脑炎患者11例,发病年龄17~68(47.18±16.20)岁,男女患者之比为5꞉6。7例为边缘性脑炎,1例为莫旺综合征,3例为获得性神经性肌强直;临床表现为癫痫发作者4例,精神行为异常、认知障碍和意识障碍各1例;脑脊液异常3例,主要表现为白细胞数及蛋白质含量轻度升高;脑电图异常5例,主要表现为慢波增多(3/5)及痫性放电(2/5);颅脑MRI异常2例,主要表现为颞叶、海马T_(2)WI及FLAIR高信号。16例抗LGI1抗体脑炎及8例抗CASPR2抗体脑炎患者使用糖皮质激素、免疫球蛋白及血浆置换等一线免疫治疗,均反应良好,其中1例抗LGI1抗体脑炎及3例抗CASPR2抗体脑炎患者同时口服吗替麦考酚酯维持治疗;采用免疫治疗的患者除2例失访外,随访期内(6~36个月)均未见明显复发。2种抗体脑炎在认知障碍、低钠血症、颅脑MRI异常上差异均有统计学意义(均P<0.05)。结论:抗LGI1抗体及抗CASPR2抗体脑炎均可表现为边缘性脑炎,抗LGI1抗体脑炎以认知障碍为主要表现,面臂肌张力障碍发作及低钠血症为特征性表现,易出现颅脑MRI异常;抗CASPR2抗体脑炎以癫痫发作为最常见临床表现,可累及周围神经系统而出现神经性肌强直、莫旺综合征且易合并肿瘤。2种抗体脑炎均对免疫治疗敏感,预后良好。

Primary structure of p-momorcharin, a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.

基于fMRI和DTI的抗LGI1抗体自身免疫性脑炎的脑功能和结构特点分析

目的:分析基于功能性磁共振成像(fMRI)和弥散张量成像(DTI)的抗富亮氨酸胶质失活蛋白1(LGI1)抗体自身免疫性脑炎的脑功能和结构特点,为理解抗LGI1抗体自身免疫性脑炎的神经病理机制提供思路。方法:选取2019年8月至2022年7月我院收治的25例抗LGI1抗体自身免疫性脑炎患者作为研究组,选取同期招募的25例无脑部疾病志愿者作为对照组。50例研究对象分别接受量表评估、fMRI和DTI检查,基于fMRI检查进行局部一致性(ReHo)分析,基于DTI检查结果对脑白质纤维结构进行分析。结果:研究组的改良Rankin量表(mRS)评分显著性高于对照组(P<0.05),简易精神状态检查量表(MMES)、蒙特利尔认知评估量表(MoCA)评分显著性低于对照组(P<0.05)。研究组受试者中,其右侧颞上回、右侧颞中回以及左侧尾状核的ReHo相比于对照组受试者出现了显著的降低变化(P<0.05)。在研究组受试者中,其前放射冠、上放射冠、后放射冠、内囊前肢、内囊的晶状体后部分、丘脑后辐射、外囊、上纵束、胼胝体压部、胼胝体体部、胼胝体膝部的向异性分数(FA)和平均弥散率(MD)相比于对照组受试者分别出现显著降低、显著升高的变化(P<0.05)。结论:抗LGI1抗体自身免疫性脑炎患者可出现记忆功能、认知功能、运动功能衰退的现象,与大脑存在广泛的脑白质纤维病变有关。

基于G_(NS)蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立

旨在建立牛流行热病毒(BEFV)感染与疫苗免疫的抗体ELISA诊断方法。在证实BEFV G_(NS)截短体与BEFV感染血清特异性反应的基础上,利用原核表达系统表达纯化G_(NS)全长蛋白并以其作为包被抗原,优化反应条件,建立基于BEFV G_(NS)的鉴别病毒感染与疫苗免疫的BEFV间接ELISA方法。结果显示:在大肠杆菌中成功表达G_(NS)蛋白,主要以包涵体的形式存在,纯化获得73 ku的重组蛋白。Western blot结果显示,纯化后的重组蛋白与BEFV感染牛血清反应原性良好,且与BEF疫苗免疫牛血清未见反应。G_(NS)抗原最佳包被浓度为0.50μg·mL^(-1),血清最佳稀释度为1∶20,酶标二抗最佳稀释度为1∶4000,阴阳性临界值为S/P值0.2069。该方法与BVDV、FMDV以及IBRV阳性血清无交叉反应,特异性良好;批内和批间变异系数均小于10%,重复性良好,可用于BEFV感染与疫苗免疫牛的鉴别诊断。

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein （CP） of tobacco mosaic virus （TMV）, which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C （1） and was independent of the salicylic acid （SA） signal pathway. We provided evidence for the SA-independent biological induction of resistance.

CASPR2/LGI1抗体双阳Isaccs综合征的临床观察

目的总结接触蛋白相关样蛋白2(contactin-associated protein 2,CASPR2)/富亮氨酸胶质瘤失活蛋白1(leucine-rich glioma inactivated 1,LGI1)抗体双阳Isaacs综合征的临床表现。方法回顾性分析首都医科大学宣武医院收治的1例CASPR2/LGI1抗体双阳Isaacs综合征患者的临床表现和诊治经过。结果本例患者病程中有肌肉颤搐、大汗、体位性低血压、窦性心动过速、小便障碍、睡眠障碍、低钠血症、体质量下降等临床表现。肌电图提示肌颤搐、束颤电位,血清CASPR2/LGI1抗体双阳性。临床表现既符合Isaacs综合征,又符合轻型Morvan综合征,予以小剂量激素治疗后症状完全缓解。结论Isaacs综合征和Morvan综合征具有相同的病因和发病机制,临床表现存在重叠,鉴别较困难,均对激素及丙球治疗效果好,可能是临床表现不同的同一种综合征。